Enhancement of laccase reactions

ABSTRACT

The present invention relates to a detergent composition, detergent additive as well as a method of oxidizing a substrate which comprises contacting the substrate with a composition which includes an enzyme selected from the group consisting of laccase, catechl oxidase, monophenol monooxygenase, and bilirubin oxidase and an enhancing agent.

This application is a divisional application of application Ser. No.08/569,101 filed Dec. 21, 1995 which is a national application ofPCT/DK94/00210 filed May 31, 1994, and claims priority of Danishapplication Serial No. 773/93 filed Jun. 29, 1993, the contents of whichare fully incorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention relates to activation of enzymes. More specifically, theinvention relates to agents capable of enhancing the activity oflaccases and related enzymes.

The invention also relates to methods of oxidizing a substrate in thepresence of a laccase enzyme, and an enhancing agent. More specifically,the invention relates to a method of bleaching of dye in solutions, to amethod of inhibiting the transfer of a textile dye from a dyed fabric toanother fabric when said fabrics are washed together in a wash liquor,to a method of bleaching lignin-containing material, in particularbleaching of pulp for paper production, to a method of treatment ofwaste water from pulp manufacturing, and to a method of enzymaticpolymerization and/or modification of lignin or lignin containingmaterial.

BACKGROUND ART

Laccases (E.C. 1.10.3.2) are enzymes that catalyse the oxidation of asubstrate (an electron or hydrogen donor) with oxygen. Such enzymes areknown from microbial, plant and animal origins, e.g. from fungi. Theyare typically copper proteins, i.e. they contain a copper atom or atomsas a prosthetic group.

Use of laccases has been suggested e.g. in bleaching of pulp for paperproduction, in treatment of waste water from pulp production, forimproved bleaching in laundry detergents, for dye transfer inhibitionduring laundering, and for lignin modification, e.g. in particle boardproduction.

The compound 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate), ABTS,supplied by Boehringer Mannheim, is a chromogenic substrate, and acommon peroxidase and phenol oxidase assay agent. These enzymes catalysethe oxidation of ABTS by hydrogen peroxide and dioxygen, respectively,producing a greenish-blue colour, which process may be monitoredphotometrically.

ABTS has been found to form a stable radical cation when oxidized by alaccase enzyme (polyphenol oxidase, EC 1.10.3.2), and has been proposedto act as a redox mediator for oxidation of non-phenolic lignin modelcompounds Bourbonnais R., Paice M. G.; FEBS Lett (1990) 267 99-102!.

Studies on demthylation and delignification of kraft pulp by a laccaseenzyme in the presence of ABTS showed that the extent of partialdemethylation by laccase was increased in the presence of ABTSBourbonnais, R. and Paice, M. G.; Appl. Microbiol. Biotechnol. (1992) 36823-827!.

Certain oxidizable substrates e.g. metal ions and phenolic compoundssuch as 7-hydroxycoumarin (7 HCm), vanillin (VAN), andp-hydroxybenzenesulfonate (pHBS), have been described as accelerators orenhancers, able to enhance bleaching reactions (cf. e.g. WO 92/18683, WO92/18687, and Kato M. and Shimizu S., Plant Cell Physiol. 1985 26 (7),pp. 1291-1301 (cf. Table 1 particular), or Saunders B. C. et al.,Peroxidase, London, 1964, p. 141 ff).

SUMMARY OF THE INVENTION

It has now surprisingly been found that organic chemical compoundsconsisting of at least two aromatic rings, of which aromatic rings atleast one ring is substituted with one or more of the following atoms:nitrogen, oxygen, and sulfur, and which aromatic rings may furthermorebe fused rings, are capable of enhancing the activity of laccaseenzymes.

Accordingly, in its first aspect, the invention provides an agent forenhancing the activity of laccase enzymes, which agent is an organicchemical compound consisting of at least two aromatic rings, of whicharomatic rings at least one ring is substituted with one or more of thefollowing atoms: nitrogen, oxygen, and sulfur; and which aromatic ringsmay furthermore be fused rings.

In a more specific aspect, the invention provides an agent for enhancingthe activity of laccase enzymes, which agent is an organic chemicalcompound of the general formula I: ##STR1##

in which general formula A represents a single bond, or one of thefollowing groups: (--CR¹¹ ═CR¹⁵ --)_(n), (--CR¹¹ ═N--N═CR¹⁵ --), (--NR¹¹--), (--CR¹² ═N--)_(n), (--NR¹¹ --CR¹² ═N--N═CR¹⁵ --), (--NR¹¹ --CR¹²═N--)_(n), (--CR¹² ═N--NR¹¹ --), (--NR¹¹ --CR¹² ═CR¹³ --), (--N═N--), inwhich groups n represents an integer of from 1 to 6; or A representscarbon, carbonyl, nitrogen, sulfur, oxygen, selenium, or phosphor, whichcarbon, phosphor and nitrogen may be unsubstituted or substituted with asubstituent group R¹¹ ;

and in which general formula the substituent groups R¹ -R¹³ and R¹⁵,which may be identical or different, independent represents any of thefollowing radicals: hydrogen, hydroxy, formyl, carboxy and esters andsalts hereof, carbamoyl, sulfo and esters and salts hereof, sulfamoyl,phospho, phosphono, phosponooxy, sulfandiyl, nitro, amino, phenyl, C₁-C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁ -C₅ -alkyl;which carbamoyl, sulfamoyl, and amino groups may furthermore beunsubstituted or substituted once or twice with a substituent group R¹⁴; and which phenyl may furthermore be unsubstituted or substituted withone or more substituent groups R¹⁴ ; and which C₁ -C₁₄ -alkyl, C₁ -C₅-alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkyl groups may besaturated or unsaturated, branched or unbranched, and may furthermore beunsubstituted or substituted with one or more substituent groups R¹⁴ ;

which substituent group R¹⁴ represents any of the following radicals:hydroxy, formyl, carboxy and esters and salts hereof, carbamoyl, sulfoand esters and salts hereof, sulfamoyl, nitro, amino, phenyl,aminoalkyl, piperidino, piperazinyl, pyrrolidino, C₁ -C₅ -alkyl, C₁ -C₅-alkoxy; which carbamoyl, sulfamoyl, and amino groups may furthermore beunsubstituted or substituted once or twice with hydroxy, C₁ -C₅ -alkyl,C₁ -C₅ -alkoxy; and which phenyl may be furthermore be substituted withone or ore of the following radicals: hydroxy, amino, formyl, carboxyand esters and salts hereof, carbamoyl, sulfo and esters and saltshereof, and sulfamoyl; and which C₁ -C₅ -alkyl, and C₁ -C₅ -alkoxygroups may furthermore be saturated or unsaturated, branched orunbranched, and may furthermore be substituted once or twice with any ofthe following radicals: hydroxy, amino, formyl, carboxy and esters andsalts hereof, carbamoyl, sulfo and esters and salts hereof, andsulfamoyl;

or in which general formula R⁵ and R⁶ may together form a group --B--,in which B represents a single bond, the group (--CH═CH--)_(n), or thegroup (--CH═N--)_(n), in which groups n represents an integer of from 1to 6; or B represents carbon, nitrogen, sulfur, oxygen, selenium, ortellurium, which carbon and nitrogen may be unsubstituted or substitutedwith a substituent group R¹⁴ as defined above;

or in which general formula two of the substituent groups R¹ -R¹⁰ maytogether form a group --C--, in which C represents any of the followinggroups: (--CHR¹¹ --N═N--), (--CH═CH--)_(n), (--CH═N--)_(n), in whichgroups n represents an integer of from 2 to 4, and in which groups R¹¹is a substituent group as defined above;

or in which general formula R⁵ and R¹², and/or R⁶ and R¹³, when n in theabove formula being 1, may together form a group --D--, in which Drepresents the groups: (--CH¹¹ --), (--NR¹¹ --), (--CR¹¹ ═CR¹⁵ --),(--CR¹¹ ═N--), (--N═CH¹¹ --), (--O--), (>C═O) or (--S--), and in whichgroups R¹¹ and R¹⁵ are substituent groups as defined above.

In another specific aspect, the invention provides an agent forenhancing the activity of a laccase enzyme, which agent is an organicchemical compound of the general formula II: ##STR2##

in which the substituent groups R¹ -R⁸, which may be identical ordifferent, independently represents any of the following radicals:hydrogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁-C₅ -alkyl; which carbamoyl, sulfamoyl, and amino groups may furthermorebe unsubstituted or substituted once or twice with a substituent groupR⁹ ; and which phenyl may further be substituted or substituted with oneor more substituent groups R⁹ ; and which C₁ -C₁₄ -alkyl, C₁ -C₅-alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkyl groups may besaturated or unsaturated, branched or unbranched, and may furthermore beunsubstituted or substituted with one or more substituent groups R⁹ ;

which substituent group R⁹ represents any of the following radicals:hydroxy, formyl, carboxy and esters and salts hereof, carbamoyl, sulfoand esters and salts hereof, sulfamoyl, nitro, amino, phenyl, C₁ -C₅-alkyl, C₁ -C₅ -alkoxy; which carbamoyl, sulfamoyl, and amino groups mayfurthermore be unsubstituted or substituted once or twice with hydroxy,C₁ -C₃ -alkyl, C₁ -C₃ -alkoxy; and which phenyl may furthermore besubstituted with one or more of the following radicals: hydroxy, amino,formyl, carboxy and esters and salts hereof, carbamoyl, sulfo and estersand salts hereof, and sulfamoyl; and which C₁ -C₅ -alkyl, and C₁ -C₅-alkoxy groups may furthermore be saturated or unsaturated branched orunbranched, and may furthermore be substituted once or twice with any ofthe following radicals: hydroxy, amino, formyl, carboxy, and esters andsalts hereof, carbamoyl, sulfo and esters and salts hereof, andsulfamoyl;

or in which general formula two of the substituent groups R¹ -R⁸ maytogether from a group --B--, in which B represents any of the followinggroups: (--N═N--), (--CH═CH--)_(n), (--CH═N--)_(n), (--N═CR⁹ --NR¹⁰ --)or (--N═N--CR⁹ --), in which groups n represents an integer of from 1 to3, R⁹ is a substituent group as defined above and R¹⁰ is defined as R⁹.

In another aspect, the invention provides a method for oxidizing asubstrate with a laccase or a laccase related enzyme, in the presence ofoxygen and in the presence of an enhancing agent of the invention.

In a more specific aspect, the invention provides a method for bleachingdye in solutions by treatment with a laccase or a laccase related enzymein the presence of oxygen in the presence of an enhancing agent of theinvention.

In another specific aspect, the invention provides a method ofinhibiting the transfer of a textile dye from a dyed fabric to anotherfabric when said fabrics are washed together in a wash liquor, themethod comprising treatment of the wash liquor, with a laccase or alaccase related enzyme in the presence of oxygen and in the presence ofan enhancing agent of the invention.

In another aspect, the invention provides a method of bleachinglignin-containing material, in particular bleaching of pulp for paperproduction, the method comprising treatment of the lignin or lignincontaining material with a laccase or a laccase related enzyme in thepresence of a source of oxygen and in the presence of an enhancing agentof the invention.

In a further aspect, the invention provides a method of enzymaticpolymerization and/or modification of lignin or lignin containingmaterial, the method comprising treatment of the lignin or lignincontaining material with a laccase or a laccase related enzyme in thepresence of a source of oxygen and in the presence of an enhancing agentof the invention.

In a yet further aspect, the invention provides a method of treatment ofwaster water, in particular waste water from pharmaceutical or chemicalindustry, e.g. waste water from dye manufacturing, from textileindustry, or from pulp manufacturing, the method comprising treatment ofthe waste water with a laccase or a laccase related enzyme in thepresence of a source of oxygen and in the presence of an enhancing agentof the invention.

In a particular aspect, the invention provides a detergent additivecapable of inhabiting the transfer of a textile dye from a dyed fabricto another fabric when said fabrics are washed together in a washliquor, the detergent additive comprising an enzyme exhibiting laccaseactivity and oxygen and an enhancing agent of the invention.

In other aspects, the invention provides detergent additives anddetergent compositions capable of inhibiting the transfer of a textiledye from a dyed fabric to another fabric when said fabrics are washedtogether in a wash liquor, the detergent composition comprising anenzyme exhibiting laccase activity and oxygen and an enhancing agent ofthe invention.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to theaccompanying drawings, in which:

FIG. 1 shows the specific formulas of some enhancing agents of theinvention (I) 6-hydroxy-2-naphtoic acid; (II) 7-methoxy-2-naphtol; (III)7-amino-2-naphthalene sulfonic acid; (IV) 5-amino-2-naphthalene sulfonicacid; (V) 1,5-diaminonaphthalene; (VI) 7-hydroxy-1,2-naphthimidazole;(VII) 10-methylphenothiazine; (VIII) 10-phenothiazine-propionic acid;(IX) N-hydroxysuccinimide-10-phenothiazine-propionate; (X) benzidine;(XI) 3,3'-dimethylbenzidine; (XII) 3,3'-dimethoxybenzidine; (XIII)3,3',5,5'-tetramethylbenzidine; (XIV) 4'-hydroxy-4-biphenylcarboxylicacid; (XV) 4-amino-4'-methoxystilbene; (XVI)4,4'-diaminostilbene-2,2'-disulfonic acid; (XVII)4,4'-diaminodiphenylamine; (XVIII) 2,7-diaminofluorene; (XIX)4,4'-dihydroxy-biphenylene; (XX) triphenylamine; (XXI)10-ethyl-4-ethyl-4-phenothiazinecarboxylic acid; (XXII)10-ethylphenothiazine; (XXIII) 10-propylphenothiazine; (XXIV)10-isopropylphenothiazine; (XXV) methyl-10-phenothiazinepropionate;(XXVI) 10-phenylphenothiazine; (XXVII) 10-allylphenothiazine; (XXVIII)10-phenoxazinepropionic acid; (XXIX)10-(3-(4-methyl-1-piperazinyl)propyl)phenothizine; (XXX)10-(2-pyrrolidinoethyl)phenothiazine; (XXXI) 10-methylphenoxazine;(XXXII) iminostilbene; (XXXIII)2-(p-aminophenyl)-6-methylbenzothiazole-7-sulfonic acid; (XXXIV)N-benzylidene-4-biphenylamine; (XXXV) 5-amino-2-naphthalenesulfonicacid; (XXXVI) 7-methoxy-2-naphtol; (XXXVII) 4,4'-dihydroxybenzophenone;(XXXVIII) N-(4-(dimethylamino)benzylidene)-p-anisidine; (XXXIX)3-methyl-2-benzothiazolinone(4-(dimethylamino)benzylidene)hydrazone;(XL) 2-acethyl-10-methylphenothiazine; (XLI)10-(2-hydroxyethyl)phenothiazine; (XLII) 10-(2-hydroxyethyl)phenoxazine;(XLIII) 10-(3-hydroxypropyl)phenothiazine; (XLIV)4,4'-dimethoxy-N-methyl-diphenylamine; (XLV) vanillin azine.

FIG. 2 shows the bleaching of Direct Blue 1 with bilirubin oxidase and10-methylphenothiazine (MPT) (□ 0 μM MPT; ♦ 2.5 μM MPT; ▪ 5.0 μM MPT; ⋄10.0 μM MPT).

DETAILED DISCLOSURE OF THE INVENTION

The Enhancing Agent

The present invention relates to the use of the chemical compounds forenhancing the activity of laccase enzymes. Accordingly, the inventionprovides an agent capable of enhancing the effect of a laccase enzyme inthe following termed enhancing agent.

The enhancing agent of the invention is an organic chemical compoundconsisting of at least two aromatic rings, of which aromatic rings atleast one ring is substituted with one or more nitrogen, oxygen, and/orsulfur atoms, and which aromatic rings may furthermore be fused rings.

In a more preferred embodiment, the enhancing agent of the invention isan organic chemical compound of the general formula I: ##STR3##

in which general formula A represents a single bond, or one of thefollowing groups: (--CR¹¹ ═CR¹⁵ --)_(n), (--CR¹¹ ═N--N═CR¹⁵ --), (--nR¹¹--), (--CR¹³ ═N--)_(n), (--NR¹¹ --CR¹² ═N--N═CR¹⁵ --), (--NR¹¹ --CR¹²═N--)_(n), (--CR¹² ═N--NR¹¹ --), (--NR¹¹ --CR¹² ═CR¹³ --), (--N═N--), inwhich groups n represents an integer of from 1 to 6; or A representscarbon, carbonyl, nitrogen, sulfur, oxygen, selenium, or phosphor, whichcarbon, phosphor and nitrogen may be unsubstituted or substituted with asubstituent group R¹¹ ;

and in which general formula the substituent groups R¹ -R¹³ and R¹⁵,which may be identical or different, independently represents any of thefollowing radicals: hydrogen, hydroxy, formyl, carboxy and esters andsalts hereof, carbamoyl, sulfo and esters and salts hereof, sulfamoyl,phospho, phosphono, phosphonooxy, sulfandiyl, nitro, amino, phenyl, C₁-C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁ -C₅ -alkyl;which carbamoyl, sulfamoyl, and amino groups may furthermore beunsubstituted or substituted once or twice with a substituent group R¹⁴; and which phenyl may furthermore be unsubstituted or substituted withone or more substituent groups R¹⁴ ; and which C₁ -C₁₄ -alkyl, C₁ -C₅-alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkyl groups may besaturated or unsaturated, branched or unbranched, and may furthermore beunsubstituted or substituted with one or more substituent groups R¹⁴ ;

which substituent group R¹⁴ represents any of the following radicals:hydroxy, formyl, carboxy and esters and salts hereof, carbamoyl, sulfoand esters and salts hereof, sulfamoyl, nitro, amino, phenyl,aminoalkyl, piperidino, piperazinyl, pyrrolidino, C₁ -C₅ -alkyl, C₁ -C₅-alkoxy; which carbamoyl, sulfamoyl, and amino groups may furthermore beunsubstituted or substituted once or twice with hydroxy, C₁ -C₅ -alkyl,C₁ -C₅ -alkoxy; and which phenyl may furthermore be substituted with oneor more of the following radicals: hydroxy, amino, formyl, carboxy andesters and salts hereof, carbamoyl, sulfo and esters and salts hereof,and sulfamoyl; and which C₁ -C₅ -alkyl, and C₁ -C₅ -alkoxy groups mayfurthermore be saturated or unsaturated, branched or unbranched, and mayfurthermore be substituted once or twice with any of the followingradicals: hydroxy, amino, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, and sulfamoyl;

or in which general formula R⁵ and R⁶ may together form a group --B--,in which B represents a single bond, the group (--CH═CH--)_(n), or thegroup (--CH═N--)_(n), in which groups n represents an integer of from 1to 6; or B represents carbon, nitrogen, sulfur, oxygen, selenium, ortellurium, which carbon and nitrogen may be unsubstituted or substitutedwith a substituent group R¹⁴ as defined above;

or in which general formula two of the substituent groups R¹ -R¹⁰ maytogether form a group --C--, in which C represents any of the followinggroups: (--CHR¹¹ --N═N--), (--CH═CH--)_(n), (--CH═N--)_(n), in whichgroups n represents an integer of from 2 to 4, and in which groups R¹¹is a substituent group as defined above;

or in which general formula R⁵ and R¹², and/or R⁶ and R¹³, when n in theabove formula being 1, may together form a group --D--, in which Drepresents the groups: (--CHR¹¹), (--NR¹¹ --), (--CR¹¹ ═CR¹⁵ --),(--CR¹¹ ═N--), (--N═CR¹¹ --), (--O--), (>C═O) or (--S--), and in whichgroups R¹¹ and R¹⁵ are substituent groups as defined above.

In particular embodiments, the enhancing agent is2-(p-aminophenyl)-6-methylbenzothiazole-7-sulfonic acid,N-(4-(dimethylamino)benzylidene)-p-anisidine,3-methyl-2-benzothiazolinone(4-(dimethylamino)benzylidene)hydrazone,vanillin azine.

In another preferred embodiment, the enhancing agent of the invention isan organic chemical compound of the general formula II: ##STR4##

in which the substituent groups R¹ -R⁸, which may be identical ordifferent, independently represents any of the following radicals:hydrogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ - alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁-C₅ -alkyl; which carbamoyl, sulfamoyl, and amino groups may furthermorebe unsubstituted or substituted once or twice with a substituent groupR⁹ ; and which phenyl may furthermore be unsubstituted or substitutedwith one or more substituent groups R⁹ ; and which C₁ -C₁₄ -alkyl, C₁-C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkyl groups maybe saturated or unsaturated, branched or unbranched, and may furthermorebe unsubstituted or substituted with one or more substituent groups R⁹ ;

which substituent groups R⁹ represents any of the following radicals:hydroxy, formyl, carboxy and esters and salts hereof, carbamoyl, sulfoand esters and salts hereof, sulfamoyl, nitro, amino, phenyl, C₁ -C₅-alkyl, C₁ -C₅ -alkoxy; which carbamoyl, sulfamoyl, and amino groups mayfurthermore be unsubstituted or substituted once or twice with hydroxy,C₁ -C₃ -alkyl, C₁ -C₃ -alkoxy; and which phenyl may furthermore besubstituted with one or more of the following radicals: hydroxy, amino,formyl, carboxy and esters and salts hereof, carbamoyl, sulfo and estersand salts hereof, and sulfamoyl; and which C₁ -C₅ -alkyl, and C₁ -C₅-alkoxy groups may furthermore be saturated or unsaturated, branched orunbranched, and may furthermore be substituted once or twice with any ofthe following radicals: hydroxy, amino, formyl, carboxy and esters andsalts hereof, carbamoyl, sulfo and esters and salts hereof, andsulfamoyl;

or in which general formula two of the substituent groups R¹ -R⁸ maytogether form a group --B--, in which B represents any of the followinggroups: (--N═N--), (--CH═CH--)_(n), (--CH═N--)_(n), (--N═CR⁹ --NR¹⁰ --)or (--N═N--CR⁹ --), in which groups n represents an integer of from 1 to3, R⁹ is a substituent group as defined above and R¹⁰ is defined as R⁹.

In a more specific embodiment, the enhancing agent of the invention isan organic chemical compound of the following formula: ##STR5##

in which formula E represents a single bond, a carbonyl group or one ofthe following groups: (--CH═CH--)_(n), (--CH═N--)_(n) or (--NR¹¹ --), inwhich n represents an integer from 1 to 2. The substituents groups R¹-R¹¹ may be identical or different, independently being one of thefollowing radicals: hydrogen, hydroxy, formyl, carboxy and esters andsalts hereof, carbamoyl, sulfo and esters and salts hereof, sulfamoyl,nitro, amino, phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅-alkyl, aryl-C₁ -C₅ -alkyl; which carbamoyl, sulfamoyl, and amino groupsmay furthermore be unsubstituted or substituted once or twice with asubstituent group R¹⁴ ; and which phenyl may furthermore beunsubstituted or substituted with one or more substituent groups R¹⁴ ;and which C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, andaryl-C₁ -C₅ -alkyl groups may be saturated or unsaturated, branched orunbranched, and may furthermore be unsubstituted or substituted with oneor more substituent groups R¹⁴ ;

which substituent group R¹⁴ represents any of the following radicals:hydroxy, formyl, carboxy and esters and salts hereof, carbamoyl, sulfoand esters and salts hereof, sulfamoyl, nitro, amino, phenyl, C₁ -C₅-alkyl, C₁ -C₅ -alkoxy; which carbamoyl, sulfamoyl, and amino groups mayfurthermore be unsubstituted or substituted once or twice with hydroxy,C₁ -C₃ -alkyl, C₁ -C₃ -alkoxy; and which phenyl may furthermore besubstituted with one or more of the following radicals: hydroxy, amino,formyl, carboxy and esters and salts hereof, carbamoyl, sulfo and estersand salts hereof, and sulfamoyl; and which C₁ -C₅ -alkyl, and C₁ -C₅-alkoxy groups may furthermore be saturated or unsaturated, branched orunbranched, and may furthermore be substituted once or twice with any ofthe following radicals: hydroxy, amino, formyl, carboxy, and esters andsalts hereof, carbamoyl, sulfo and esters and salts hereof, andsulfamoyl;

or in which specific formula two of the substituent groups R⁵ and R⁶ maytogether form a group --B--, in which B represents the groups:('CH═N--)_(n), (--CH═CH--) or (--CHR¹⁴ --) in which groups n representsan integer of from 1 to 2 and R¹⁴ is a substituent group as definedabove.

In particular embodiments, the enhancing agent is4-amino-4'-methoxystilbene, 4,4'-diaminostilbene-2,2'-disulfonic acid,iminostilbene, 4,4'-dihydroxybenzophenone,N-benzylidene-4-biphenylamine, 4,4'-diaminodiphenylamine,4,4'-dimethoxy-N-methyl-diphenylamine, 2,7-diaminofluorene,triphenylamine.

In another specific embodiment, the enhancing agent may be described bythe following formula: ##STR6##

in which formula X represents one of the following groups: (--O--), (13S--), (--NR¹⁵ --), (--CHR¹⁵ --), (>C═O), (--CH═CH--), (--CH═N--) and thesubstituent groups R¹ -R⁹ and R¹⁵, which may be identical or different,independently represents any of the following radicals: hydrogen,hydroxy, formyl, carboxy and esters and salts hereof, carbamoyl, sulfoand esters and salts hereof, sulfamoyl, nitro, amino, phenyl, C₁ -C₁₄-alkyl; C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁ -C₅ -alkyl;which carbamoyl, sulfamoyl, and amino groups may furthermore beunsubstituted or substituted once or twice with a substituent group R¹⁰; and which one or more substituent groups R¹⁰ ; and which C₁ -C₁₄-alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkylgroups may be saturated or unsaturated, branched or unbranched, and mayfurthermore be unsubstituted or substituted with one or more substituentgroups R¹⁰ ;

which substituent group R¹⁰ represents any of the following radicals:hydroxy, formyl, carboxy and esters and salts hereof, carbamoyl, sulfoand esters and salts hereof, sulfamoyl, nitro, amino, phenyl,aminoalkyl, piperidino, piperazinyl, pyrrolidino, C₁ -C₅ -alkyl, C₁ -C₅-alkoxy; which carbamoyl, sulfamoyl, and amino groups may furthermore beunsubstituted or substituted once or twice with hydroxy, C₁ -C₅ -alkyl,C₁ -C₅ alkoxy; and which phenyl may furthermore be substituted with oneor more of the following radicals: hydroxy, amino, formyl, carboxy andesters and salts hereof, carbamoyl, sulfo and esters and salts hereof,and sulfamoyl; and which C₁ -C₅ -alkyl, and C₁ -C₅ -alkoxy groups mayfurthermore be saturated or unsaturated, branched or unbranched, and mayfurthermore be substituted once or twice with any of the followingradicals: hydroxy, amino, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, and sulfamoyl;

or in which general formula tow of the substituent groups R¹ -R⁸ maytogether form a group --B--, in which B represents any of the followingthe groups: (--CHR¹⁰ --N═N--), (--CH═CH--)_(n), ('CH═N--)_(n) or(--N═CR¹⁰ --NR¹¹ --), in which groups n represents an integer of from 1to 3, R¹⁰ is a substituent group as defined above and R¹¹ is defined asR¹⁰.

In particular embodiments, the enhancing agent is10-methylphenothiazine, 10-phenothiazine-propionic acid,N-hydroxysuccinimide-10-phenothiazine-propionate or10-ethyl-4phenothiazine-carboxylic acid, 10-ethylphenothiazine,10-propylphenothiazine, 10-isopropylphenothiazine,methyl-10phenothiazinepropionate, 10-phenylphenothiazine,10-allylphenothiazine,10-(3-(4-methyl-1-piperazinyl)propyl)phenothiazine,(10-(2-pyrrolidinoethyl)phenothiazine, 2-acetyl-10-methyl-phenothiazine,4-carboxy-10-phenothiazine, 10-methyl-phenoxazine, 10-ethylphenoxazine,10-phenoxazine-propionic acid, 4-carboxy-10-phenoxazine-propionic acid,10-(2-hydroxyethyl)phenothiazine, 10-(2-hydroxyethyl)phenoxazine or10-(3-hydroxypropyl)phenothiazine.

In another specific embodiment, enhancing agent is biphenyl derivativeof the following formula: ##STR7##

in which the substituent groups R¹ -R¹⁰, which may be identical ordifferent, independently represents any of the following radicals:hydrogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁-C₅ -alkyl; which carbamoyl, sulfamoyl, and amino groups may furthermorebe unsubstituted or substituted once or twice with a substituent groupR¹¹ ; and which phenyl may furthermore be unsubstituted or substitutedwith one or more substituent groups R¹¹ ; and which C₁ -C₁₄ alkyl, C₁-C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ alkyl groups may besaturated or unsaturated, branched or unbranched, and may be furthermorebe unsubstituted or substituted with one or more substituent groups R¹¹;

which substituent group R¹¹ represents any of the following radicals:hydroxy, formyl, carboxy and esters and salts hereof, carbamoyl, sulfoand esters and salts hereof, sulfamoyl, nitro, amino, phenyl, C₁ -C₅-alkyl, C₁ -C₅ -alkoxy; which carbamoyl, sulfamoyl, and amino groups mayfurthermore be unsubstituted or substituted once or twice with hydroxy,C₁ -C₃ -alkyl, C₁ -C₃ -alkoxy; and which phenyl may furthermore besubstituted with one or more of the following radicals: hydroxy, amino,formyl, carboxy and esters and salts hereof, carbamoyl, sulfo and estersand salts hereof, and sulfamoyl; and which C₁ -C₅ -alkyl, and C₁ -C₅-alkoxy groups may furthermore be saturated or unsaturated, branched orunbranched, and may furthermore be substituted once or twice with any ofthe following radicals: hydroxy, amino, formyl, carboxy and esters andsalts hereof, carbamoyl, sulfo and esters and salts hereof, andsulfamoyl,

In particular embodiments, the enhancing agent is benzidine,3,3'-dimethylbenzidine, 3,3'-dimethoxybenzidine,3,3',5,5'-tetramethylbenzidine, 4'-hydroxy-4-biphenylcarboxylic acid, or4,4'-dihydroxybiphenylene.

In another specific embodiment, the enhancing agent is a naphthalenederivative of the following formula: ##STR8##

in which the substituent groups R¹ -R⁸, which may be identical ordifferent, independently represents any of the following radicals:hydrogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁-C₅ -alkyl; which carbamoyl, sulfamoyl, and amino groups may furthermorebe unsubstituted or substituted once or twice with a substituent groupR⁹ ; and which phenyl may furthermore be unsubstituted or substitutedwith one or more substituent groups R⁹ ; and which C₁ -C₁₄ -alkyl, C₁-C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkyl groups maybe saturated or unsaturated, branched or unbranched, and may furthermorebe unsubstituted or substituted with one or more substituent groups R⁹ ;

which substituent group R⁹ represents any of the following radicals:hydroxy, formyl, carboxy and esters and salts hereof, carbamoyl, sulfoand esters and salts hereof, sulfamoyl, nitro, amino, phenyl, C₁ -C₅-alkyl, C₁ -C₅ -alkoxy; which carbamoyl, sulfamoyl, and amino groups mayfurthermore be unsubstituted or substituted once or twice with hydroxy,C₁ -C₃ -alkyl, C₁ -C₃ -alkoxy; and which phenyl may furthermore besubstituted with one or more of the following radicals: hydroxy, amino,formyl, carboxy and esters and salts hereof, carbamoyl, sulfo and estersand salts hereof, and sulfamoyl, and which C₁ -C₅ -alkyl, and C₁ -C₅-alkoxy groups may furthermore be saturated or unsaturated, branched orunbranched, and may furthermore be substituted once or twice with any ofthe following radicals: hydroxy, amino, formyl, carboxy and esters andsalts hereof, carbamoyl, sulfo and esters and salts hereof, andsulfamoyl;

or in which general formula two of the substituent groups R¹ -R⁸ maytogether form a group --B--, in which B represents any of the followinggroups: (--N═N--), (--CH═CH--)_(n), (--CH═N--)_(n), (--N═CR⁹ --NR¹⁰ --)or (--N═N--CR⁹ --), in which groups n represents an integer of from 1 to3, R⁹ is substituent group as defined above and R¹⁰ is defined as R⁹.

In particular embodiments, the enhancing agent is 6-hydroxy-2-naphtoicacid, 7-methoxy-2-naphtol, 7-amino-2-naphthalene sulfonic acid,5-amino-2-naphthalene sulfonic acid, 1,5-diaminonaphthalene,7-hydroxy-1,2-naphthimidazole, 5-amino-2-naphthalenesulfonic acid, or7-methoxy-2-naphtol.

The enhancing agent of the invention may be in free form or in the formof an addition salt.

Methods of Oxidizing a Substrate

In another aspect, the invention provides a method of oxidizing asubstrate with a laccase enzyme and in the presence of an enhancingagent.

The enhancing agent may be present in free form or in the form of anaddition salt.

The enhancing agent may be present in concentrations of from 0.01 to 500μM, more preferred 0.1 to 250 μM, most preferred 0.5 to 100 μM.

Molecular oxygen from the atmosphere will usually be present insufficient quantity.

Laccase and Related Enzymes

The enhancing agents of the invention are agents capable of enhancingthe activity of laccases and laccase related enzymes. In the context ofthis invention, laccases and laccase related enzymes contemplate anylaccase enzyme comprised by the enzyme classification EC 1.10.3.2, anychatechol oxidase enzyme comprised by the enzyme classification EC1.10.3.1, any bilirubin oxidase enzyme comprised by the enzymeclassification EC 1.3.3.5 or any monophenol monooxygenase enzymecomprised by the enzyme classification EC 1.14.99.1.

Preferably, the laccase employed in the method of the invention isderived from a fungi such as Trametes, e.g. T. villosa or T. versicolor,Collybia, Fomes, Lentinus, Pleurotous, Rhizoctonia, e.g. R. solani,Aspergillus, Neurospora, Podospora, Phlebia, e.g. P. radiata (WO92/01046), Coriolus, e.g. C. hirsitus (JP 2-238885), Myceliophthora,e.g. M. thermophila or Botrytis. Bilirubin oxidase may preferably bederived from Myrothecium, e.g. M. verrucaria.

The laccase or the laccase related enzyme may furthermore be one whichis producible by a method comprising cultivating a host cell transformedwith a recombinant DNA vector which carries a DNA sequence encoding saidlaccase as well as DNA sequences encoding functions permitting theexpression of the DNA sequence encoding the laccase, in a culture mediumunder conditions permitting the expression of the laccase enzyme, andrecovering the laccase from the culture.

Determination of Laccase Activity (LACU)

Laccase activity is determined from the oxidation of syringaldazin underaerobic conditions. The violet colour produced is photometered at 530nm. The analytical conditions are 19 μM syringaldazin, 23.2 mM acetatebuffer, pH 5.5, 30° C., 1 min. reaction time.

1 laccase unit (LACU) is the amount of enzyme that catalyses theconversion of 1.0 μmole syringaldazin per minute at those conditions.

Determination of Bilirubin Oxidase Activity (Sigma Units)

1 bilirubin oxidase Sigma unit is the amount of enzyme that will oxidize1.0 μmole of bilirubin per minute at pH 8.4 and 30° C.

Industrial Applications

In a preferred embodiment, the method of the invention finds applicationfor bleaching of dye in solutions.

In another embodiment, the method of the invention finds application fordye transfer inhibition, e.g. for treatment of dyed textiles (cf. e.g.WO 92/18687) or during laundering (cf. e.g. WO 91/05839).

Accordingly, in a specific embodiment, the invention provides a methodinhibiting the transfer of a textile dye from a dyed fabric to anotherfabric when said fabrics are washed together in a wash liquor, themethod comprising treatment of the wash liquor with a laccase or alaccase related enzyme in the presence of an enhancing agent. Thetextile dye may be a synthetic dye such as an azo dye, or a natural ornature-identical dye.

In a third embodiment, the method of the invention finds application inbleaching of pulp for paper production.

Accordingly, the invention provides a method for bleaching oflignin-containing material, in particular bleaching of pulp for paperproduction, which method comprises treatment of the lignin or lignincontaining material with a laccase or a laccase related enzyme in thepresence of an enhancing agent of the invention.

In a fourth embodiment, the method of the invention finds applicationfor lignin modification, e.g. in the manufacture of wood composites,e.g. wood fibre materials such as chipboards, fibre boards, or particleboards, or in the manufacture of laminated wood products, such aslaminated beams and plywood.

Accordingly, the invention provides a method for enzymaticpolymerization and/or modification of lignin or lignin containingmaterial, which method comprises treatment of the lignin or lignincontaining material with a laccase or a laccase related enzyme in thepresence of an enhancing agent of the invention.

In a fifth embodiment, the method of the invention finds application intreatment of waste water e.g. waste water from the chemical orpharmaceutical industry, from dye manufacturing, from dye-works, fromthe textile industry, or from pulp production (cf. e.g. U.S. Pat. No.4,623,465, or JP-A-2-31887).

In a more specific aspect, the invention provides a method for treatmentof waste water from dye manufacturing, from dye-works, from textileindustry, or from pulp manufacturing, the method comprising treatment ofthe waste water with a laccase or a laccase related enzyme in thepresence of an enhancing agent of the invention.

Detergent Compositions

According to the invention, the enhancing agent may be added as acomponent of a detergent composition. As such, it may be included in thedetergent composition in the form of a detergent additive. The detergentcomposition as well as the detergent additive may additionally compriseone or more other enzymes conventionally used in detergents, such asproteases, lipases, amylases, cutinases, cellulases and peroxidases.

In a specific aspect, the invention provides a detergent additive. Theenzymes may be included in a detergent composition by adding separateadditives containing one or more enzymes, or by adding a combinedadditive comprising all of these enzymes. A detergent additive of theinvention, i.e. a separated additive or a combined additive, can beformulated e.g. as granulates, liquids, slurries, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids,slurries, or protected enzymes.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 (both to Novo Industri A/S) an mayoptionally be coated by methods known in the art. Examples of waxycoating materials are poly(ethylene oxide) products (polyethyleneglycol,PEG) with means molar weights of 1000 to 20000; ethoxylated nonylphenolshaving from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols inwhich the alcohol contains from 12 to 20 carbon atoms and in which thereare 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; andmono- and di- and triglycerides of fatty acids. Examples of film-formingcoating materials suitable for application by fluid bed techniques aregiven in patent GB 1483591. Liquid enzyme preparations may, forinstance, be stabilized by adding a polyol such as propylene glycol, asugar or sugar alcohol, lactic acid or boric acid according toestablished methods. Other enzyme stabilizers are well known in the art.Protected enzymes may be prepared according to the method disclosed inEP 238,216.

The detergent composition of the invention may be in any convenientform, e.g. as powder, granules, paste or liquid. A liquid detergent maybe aqueous, typically containing up to 70% water and 0-30% organicsolvent, or nonaqueous.

The detergent composition comprises one or more surfactants, each ofwhich may be anionic, nonionic, cationic, or zwitterionic. The detergentwill usually contain 0-50% of anionic surfactant such as linearalkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate(fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES),secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters,alkyl- or alkenylsuccinic acid or soap. It may also contain 0-40% ofnonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylatedalcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. asdescribed in WO 92/06154).

The detergent composition may additionally comprise one or more olderenzymes, such as amylase, lipases, cutinases, proteases, cellulases anperoxidases.

The detergent may contain 1-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate, citrate,nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).The detergent may also be unbuilt, i.e. essentially free of detergentbuilder.

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),olyethyleneglycol (PEG), poly(vinyl alcohol) PVA), polycarboxylates suchas polyacrylates, maleic/acrylic acid copolymers and laurylmethacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂ O₂source such as perborate or pecarbonate which may be combined with aperacid-forming bleach activator such as teraacetylethylenediamine(TAED) or nonnoyloxybenzenesulfonate (NOBS). Alternatively, thebleaching system may comprise peroxyacids of e.g. the amide, imide, orsulfone type.

The enzymes of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g. a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative as e.g. an aromatic borate ester,and the composition may be formulated as described in e.g. WO 92/19709and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g. fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners, orperfume.

The pH (measured in aqueous solution at use concentration) will usuallybe neutral or alkaline, e.g. 7-11.

Particular forms of detergent compositions within the scope of theinvention include:

1) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________    linear alkylbenzenesulfonate                             7-12%    (calculated as acid)    alcohol ethoxysulfate    1-4%    (e. g. C.sub.12-18 alcohol, 1-2 EO) or    alkyl sulfate (e.g. C.sub.16-18)    alcohol ethoxylate       5-9%    (e.g. C.sub.14-15 alcohol, 7 EO)    sodium carbonate (as Na.sub.2 CO.sub.3)                             14-20%    soluble silicate (as Na.sub.2 O,2SiO.sub.2)                             2-6%    zeolite (as NaAlSiO.sub.4)                             15-22%    sodium sulfate (as Na.sub.2 SO.sub.4)                             0-6%    sodium citrate/citric acid                             0-15%    (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7 /C.sub.6 H.sub.8 O.sub.7)    sodium perborate (as NaBO.sub.3 · H.sub.2 O)                             11-18%    TAED                     2-6%    carboxymethylcellulose   0-2%    polymers (e.g. maleic/acrylic acid                             0-3%    copolymer, PVP, PEG)    enzymes                  0-5%    minor ingredients (e.g. suds                             0-5%    suppressors, perfume, optical    brightener, photobleach)    ______________________________________

2) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________    linear alkylbenzenesulfonate                               6-11%    (calculated as acid)    alcohol ethoxysulfate      1-3%    (e.g. C.sub.12-18 alcohol, 1-2 EO)    or alkyl sulfate (e.g. C.sub.16-18)    alcohol ethoxylate         5-9%    (e.g. C.sub.14-15 alcohol, 7 EO)    sodium carbonate (as Na.sub.2 CO.sub.3)                               15-21%    soluble silicate (as Na.sub.2 O,2SiO.sub.2)                               1-4%    zeolite (as NaAlSiO.sub.4) 24-34%    sodium sulfate (as Na.sub.2 SO.sub.4)                               4-10%    sodium citrate/citric acid 0-15%    (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7 /C.sub.6 H.sub.8 O.sub.7)    carboxymethylcellulose     0-2%    polymers (e.g. maleic/acrylic acid copolymer,                               1-6%    PVP, PEG)    enzymes                    0-5%    minor ingredients          0-5%    (e.g. suds suppressors, perfume)    ______________________________________

3) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________    linear alkylbenzenesulfonate                               5-9%    (calculated as acid)    alcohol ethoxylate         7-14%    (e.g. C.sub.12-15 alcohol, 7 EO)    soap as fatty acid         1-3%    (e.g. C.sub.16-22)    sodium carbonate (as Na.sub.2 CO.sub.3)                               10-17%    soluble silicate (as Na.sub.2 O,2SiO.sub.2)                               3-9%    zeolite (as NaAlSiO.sub.4) 23-33%    sodium sulfate (as Na.sub.2 SO.sub.4)                               0-4%    sodium perborate (as NaBO.sub.3 · H.sub.2 O)                               8-16%    TAED                       2-8%    phosphonate (e.g. EDTMPA)  0-1%    carboxymethylcellulose     0-2%    polymers (e.g. maleic/acrylic acid ccpolymer,                               1-3%    PVP, PEG)    enzymes                    0-5%    minor ingredients (e.g. suds suppressors,                               0-5%    perfume, optical brightener)    ______________________________________

4) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________    linear alkylbenzenesulfonate                               8-12%    (calculated as acid)    alcohol ethoxylate         10-25%    (e.g. C.sub.12-15 alcohol, 7 EO)    sodium carbonate (as Na.sub.2 CO.sub.3)                               14-22%    soluble silicate (as Na.sub.2 O,2SiO.sub.2)                               1-5%    zeolite (as NaAlSiO.sub.4) 25-35%    sodium sulfate (as Na.sub.2 SO.sub.4)                               0-10%    carboxymethylcellulose     0-2%    polymers (e.g. maleic/acrylic acid copolymer,                               1-3%    PVP, PEG)    enzymes                    0-5%    minor ingredients (e.g. suds suppressors,                               0-5%    perfume)    ______________________________________

5) An aqueous liquid detergent composition comprising

    ______________________________________    linear alkylbenzenesulfonate                             15-21%    (calculated as acid)    alcohol ethoxylate       12-18%    (e.g. C.sub.12-15 alcohol, 7 EO or    C.sub.12-15 alcohol, 5 EO)    soap as fatty acid (e.g. oleic acid)                             3-13%    alkenylsuccinic acid (C.sub.12-14)                             0-13%    aminoethanol             8-18%    citric acid              2-8%    phosphanate              0-3%    polymers (e.g. PVP, PEG) 0-3%    borate (as B.sub.4 O.sub.7)                             0-2%    ethanol                  0-3%    propylene glycol         8-14%    enzymes                  0-5%    minor ingredients        0-5%    (e.g. dispersants, suds suppressors,    perfume, optical brightener)    ______________________________________

6) An aqueous structured liquid detergent composition comprising

    ______________________________________    linear alkylbenzenesulfonate                               15-21%    (calculated as acid)    alcohol ethoxylate         3-9%    (e.g. C.sub.12-15 alcohol, 7 EO    or C.sub.12-15 alcohol, 5 EO)    soap as fatty acid (e.g. oleic acid)                               3-10%    zeolite (as NaAlSiO.sub.4) 14-22%    potassium citrate          9-18%    borate (as B.sub.4 O.sub.7)                               0-2%    carboxymethylcellulose     0-2%    polymers (e.g PEG, PVP)    0-3%    anchoring polymers as      0-3%    e.g. lauryl methacrylate/acrylic acid copolymer;    molar ratio 25:1; MW 3800    glycerol                   0-5%    enzymes                    0-5%    minor ingredients          0-5%    (e.g. dispersants, suds suppressors, perfume,    optical brighteners)    ______________________________________

7) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________    fatty alcohol sulfate      5-10%    ethoxylated fatty acid monoethanolamide                               3-9%    soap as fatty acid         0-3%    sodium carbonate (as Na.sub.2 CO.sub.3)                               5-10%    soluble silicate (as Na.sub.2 O,2SiO.sub.2)                               1-4%    zeolite (as NaAlSiO.sub.4) 20-40%    sodium sulfate (as Na.sub.2 SO.sub.4)                               2-8%    sodium perborate (as NaBO.sub.3 · H.sub.2 O)                               12-18%    TAED                       2-7%    polymers (e.g. maleic/acrylic acid copolymer,                               1-5%    PEG)    enzymes                    0-5%    minor ingredients (e.g. optical brightener,                               0-5%    suds suppressors, perfume)    ______________________________________

8) A detergent composition formulated as a granulate comprising

    ______________________________________    linear alkylbenzenesulfonate                              8-14%    (calculated as acid)    ethoxylated fatty acid monoethanolamide                              5-11%    soap as fatty acid        0-3%    sodium carbonate (as Na.sub.2 CO.sub.3)                              4-10%    soluble silicate (as Na.sub.2 O,2SiO.sub.2)                              1-4%    zeolite (as NaAlSiO.sub.4)                              30-50%    sodium sulfate (as Na.sub.2 SO.sub.4)                              3-11%    sodium citrate (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7)                              5-12%    polymers (e.g. PVP,       1-5%    maleic/acrylic acid copolymer, PEG)    enzymes                   0-5%    minor ingredients (e.g. suds suppressors,                              0-5%    perfume)    ______________________________________

9) A detergent composition formulated as a granulate comprising

    ______________________________________    linear alkylbenzenesulfonate                              6-12%    (calculated as acid)    nonionic surfactant,      1-4%    soap as fatty acid        2-6%    sodium carbonate (as Na.sub.2 CO.sub.3)                              14-22%    zeolite (as NaAlSiO.sub.4)                              18-32%    sodium sulfate (as Na.sub.2 SO.sub.4)                              5-20%    sodium citrate (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7)                              3-8%    sodium perborate (as NaBO.sub.3 · H.sub.2 O)                              4-9%    bleach activator (e.g. NOBS or TAED)                              1-5%    carboxymethylcellulose    0-2%    polymers (e.g. polycarboxylate or PEG)                              1-5%    enzymes                   0-5%    minor ingredients         0-5%    (e.g. optical brigntener, perfume)    ______________________________________

10) An aqueous liquid detergent composition comprising

    ______________________________________    linear alkylbenzensulfonate                               15-23%    (calculated as acid)    alcohol ethoxysulfate      8-15%    (e.g. C.sub.12-15 alcohol, 2-3 EO)    alcohol ethoxylate         3-9%    (e.g. C.sub.12-15 alcohol, 7 EO    or C.sub.12-15 alcohol, 5 EO)    soap as fatty acid (e.g. lauric acid)                               0-3%    aminoethanol               1-5%    sodium citrate             5-10%    hydrotrope (e.g. sodium toluenesulfonate)                               2-6%    borate (as B.sub.4 O.sub.7)                               0-2%    carboxymethylcellulose     0-1%    ethanol                    1-3%    propylene glycol           2-5%    enzymes                    0-5%    minor ingredients (e.g. polymers, dispersants,                               0-5%    perfume, optical brighteners)    ______________________________________

11) An aqueous liquid detergent composition comprising

    ______________________________________    linear alkylbenzenesulfonate                              20-32%    (calculated as acid)    alcohol ethoxylate        6-12%    (e.g. C.sub.12-15 alcohol, 7 EO    or C.sub.12-15 alcohol, 5 EO)    aminoethanol              2-6%    citric acid               8-14%    borate (as B.sub.4 O.sub.7)                              1-3%    polymer (e.g. maleic/acrylic acid copolymer,                              0-3%    anchoring polymers as e.g.    lauryl methacrylate/acrylic acid    copolymer and CMC)    glycerol                  3-8%    enzymes                   0-5%    minor ingredients (e.g. hydrotropes,                              0-5%    dispersants, perfume, optical brighteners)    ______________________________________

12) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________    anionic surfactant (linear                              25-40%    alkylbenzenesulfonate, alkyl sulfate, alpha-    olefinsulfonate, alpha-sulfo fatty acid    methyl esters, alkanesulfonates, soap)    nonionic surfactant       1-10%    (e.g. alcohol ethoxylate)    sodium carbonate (as Na.sub.2 CO.sub.3)                              8-25%    soluble silicates (as Na.sub.2 O, 2SiO.sub.2)                              5-15%    sodium sulfate (as Na.sub.2 SO.sub.4)                              0-5%    zeolite (as NaAlSiO.sub.4)                              15-28%    sodium perborate (as NaBO.sub.3 · 4H.sub.2 O)                              0-20%    bleach activator (TAED or NOBS)                              0-5%    enzymes                   0-5%    minor ingredients         0-3%    (e.g. perfume, optical brighteners)    ______________________________________

13) Detergent formulations as described in 1)-12) where the content oflinear alkylbenzenesulfonate--or a part of it--is substituted by alkylsulfate (C₁₂ -C₁₈).

14) Detergent formulations as described in 1)-13) which contain astabilized or encapsulated peracid either as an additional component oras a substitute for already specified bleach systems.

15) Detergent compositions as described in 3), 7), 9) and 12) where thecontent of perborate is substituted with percarbonate.

16) Detergent composition formulated as a nonaqueous detergent liquidcomprising a liquid nonionic surfactant as e.g. linear alkoxylatedprimary alcohol, a builder system (e.g. phosphate), enzyme and alkali.The detergent may also comprise anionic surfactant and/or a bleachsystem.

The following examples further illustrate the present invention, andthey are not intended to be in any way limiting to the scope of theinvention as claimed.

EXAMPLE 1

Bleaching of Direct Blue 1 with Bilirubin Oxidase and10-Methylphenothiazine

The bleaching of Direct Blue 1 (DB1) by bilirubin oxidase obtained fromSigma with and without 10-methyl-phenothiazine (MPT) is presented in theattached FIG. 2.

The following conditions were used:

    ______________________________________                    Final concentrations    ______________________________________    Buffer            50 mM phosphate, pH 7    MPT               0, 2.5, 5, or 10 μM    DB1               ≈ Abs.sub.605nm = 0.6    Bilirubin oxidase 0.027 Sigma units/ml    ______________________________________

The experiment was started by the addition of enzyme, and the absorbanceat 605 nm was monitored for 5 minutes. The temperature was 25° C.

No attempt to maintain a constant concentration of dissolved oxygen wasmade.

As evidenced by FIG. 2, bleaching is enhanced by increasingconcentrations of the enhancer of the invention.

EXAMPLE 2

Bleaching of Direct Blue 1

Bilirubin Oxidase

Myrothecium verrucaria bilirubin oxidase was purchased from Sigma(B-0390). A stock solution was made containing 27 Sigma units/ml, whichwas stored in the freezer until use.

Laccase Obtained from Trametes villosa

800 ml culture broth of Trametes villosa, CBS 678.70, was filtered withfilter aid to give a clear filtrate, which was concentrated and washedby ultrafiltration on a membrane with a cut-off of 6-8 kDa. One mlsamples of concentrated preparation was applied onto a Q-Sepharose HPcolumn (Pharmacia, Sweden) equilibrated with 0.1M fosfate pH 7, and thelaccase was eluted with a flat NaCl gradient around 0.25M. Fractionswith laccase activity from 10 runs were pooled and concentrated byultrafiltration to an activity of 500 LACU/ml.

Enhancers

Chemicals were obtained from Sigma-Aldrich, Janssen Chimica, Kodak,Tokyo Kasai Organic Chemicals or Daiichi Pure Chemicals Co. N-methylatedderivatives of phenothiazine and phenoxazine were made by methylationwith methyliodide as described by Cornel Bodea and Ioan Silberg in"Recent Advances in the Chemistry of Phenothiazines"(Advances inheterocyclic chemistry, 1968, Vol. 9, pp. 321-460); B. Cardillo & G.Casnati in Tetrahedron, 1967, Vol. 23, p. 3771. Phenothiazine andphenoxazine propionic acids may be prepared as described in J. Org.Chem. 15, 1950, pp. 1125-1130. Hydroxyethyl and hydroxypropylderivatives of phenothiazine and phenoxazine may be prepared asdescribed by G. Cauquil in Bullentin de la Society Chemique de France,1960, p. 1049.

Due to low solubility some of the enhancers were dissolved in a smallvolume of ethanol before dilution in water.

The following conditions were used:

    ______________________________________                        Final concentration    ______________________________________    400 μl 50 mM Britton-Robinson buffer,                          20 mM    pH 5.5 and pH 7.0 respectively,    200 μl DB1 - 3.0 Abs. Units (610 nm)                          0.6 (A.sub.610nm)    200 μl 50 μM enhancer                          10 μM    200 μl Enzyme    ______________________________________

Reagents were mixed in a 1 cm thermostated cuvette at 30° C. and thebleaching was started by addition of enzyme.

The bleaching was detected spectrophotometrically at 610 nm, which isthe absorption peak of DB1. After 5 sec. bleaching was followed for 4minutes.

From the results presented in Table 1-2, below, it appears that addingenhancers of the invention a much faster bleaching of the dye can beobtained compared to the experiment without enhancer. Enzyme dosagesgiven are in the final incubation mixture.

                  TABLE 1    ______________________________________    Bleaching of Direct Blue 1 with Trametes villosa laccase,    obtained as described above, at pH 5.5 (0.1 LACU/ml) and pH 7.0    (1 LACU/ml).                    DB1 bleaching in 4 minutes                    (-.increment.mAbs/4 min)    Enhancer          pH 5.5  pH 7.0    ______________________________________    No enhancer       0       0    10-Phenothiazine- 387     182    propionic acid    10-Methyl-        222     22    phenothiazine    4'-Hydroxy-    4-biphenyl-       46      34    carboxylic acid    4,4'-    Diaminostilbene-    2,2'-disulfonic   3.5     6    acid    6-Hydroxy-2-      12      20    naphthoic acid    10-Methylphenoxa- 364     296    zine    10-Phenoxazinepro-                      364     268    pionic acid    10-Ethyl-4-pheno-    thiazinepropionic 411     175    acid    10-(2-Hydroxyethyl)-                      373     184    phenothiazine    10-(2-Hydroxyethyl)-                      429     192    phenoxazine    10-(3-Hydroxypropyl)-                      428     219    phenothiazine    4,4'-Dimethoxy-N- 210     113    methyl-diphenylamine    Vanillin azine    118     5    TABLE 2    Bleaching of Direct Blue 1 with Myrothecium verrucaria bili-    rubin oxidase, obtained as described above, at pH 5.5 (0.00675    Sigma units/ml) and pH 7.0 (0.0675 Sigma units/ml).                    DB1 bleaching in 4 minutes                    (-.increment.mAbs/4 min)    Enhancer          pH 5.5  pH 7.0    No enhancer       68      207    10-Phenothiazine- 95      244    propionic acid    10-Methyl-        76      250    phenothiazine    10-Methylphenoxa- 143     374    zine    10-Phenoxazinepro-                      96      347    pionic acid    10-Ethyl-4-pheno- 103     293    thiazinepropionic    acid    ______________________________________

What is claimed:
 1. A method of oxidizing a substrate, comprisingtreating the substrate with an enzyme selected from the group consistingof laccase, catechol oxidase, monophenol monooxygenase and bilirubinoxidase and an enhancing agent of the following formula: ##STR9##wherein E is a single bond, a carbonyl group or one of the followinggroups: (--CH═CH--)_(n), (--CH═N--)_(n) or (--NR¹¹ --), in which nrepresents an integer from 1 to 2; andR¹ -R¹¹ may be identical ordifferent and independently are one of the following radicals: hydrogen,hydroxy, formyl, carboxy or an ester or salt thereof, carbamoyl, sulfoor an ester or salt thereof, sulfamoyl, phospho, phosphono, phosphonooxyor an ester or salt thereof, nitro, amino, phenyl, C₁ -C₁₄ -alkyl, C₁-C₅ - alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁ -C₅ -alkyl; wherein thecarbamoyl, sulfamoyl, and amino groups may be unsubstituted orsubstituted once or twice with a substituent group R¹⁴ ; the phenylgroup may be unsubstituted or substituted with one or more substituentgroups R¹⁴ ; and the C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅-alkyl, and aryl-C₁ -C₅ -alkyl groups may be saturated or unsaturated,branched or unbranched, and unsubstituted or substituted with one ormore substituent groups R¹⁴ ; wherein R¹⁴ is any of the followingradicals; hydroxy, formyl, carboxy or an ester or salt thereof,carbamoyl, sulfo or an ester or salt thereof, sulfamoyl, phospho,phosphono, phosphonooxy or an ester or salt thereof, nitro, amino,phenyl, C₁ -C₅ -alkyl, C₁ -C₅ -alkoxy; wherein the carbamoyl, sulfamoyl,and amino groups may be unsubstituted or substituted once or twice withhydroxy, C₁ -C₃ -alkyl, C₁ -C₃ -alkoxy; the phenyl group may beunsubstituted or substituted with one or more of the following radicals:hydroxy, amino, formyl, carboxy or an ester or salt thereof, carbamoyl,sulfo or an ester or salt thereof, or sulfamoyl; and the C₁ -C₅ -alkyland C₁ -C₅ -alkoxy groups may be saturated or unsaturated, branched orunbranched, and unsubstituted or substituted once or twice with any ofthe following radicals: hydroxy, amino, formyl, carboxy or an ester orsalt thereof, carbamoyl, sulfo or an ester or salt thereof, orsulfamoyl; or in which R⁵ and R⁶ together form a group --B--, in which Bis (--CH═N--)_(m), (--CH═CH--) or (--CHR¹⁴ --) in which m is 1 or 2 andR¹⁴ is as defined above.
 2. The method according to claim 1, in whichthe enzyme is a laccase.
 3. The method according to claim 1, in whichthe enhancing agent is 4-amino-4'-methoxystilbene,4,4'-diaminostilbene-2,2'-disulfonic acid, iminostilbene,4,4'-dihydroxybenzophenone,N-benzylidene-4-biphenylamine,4,4'-diaminodiphenylamine,4,4'-dimethoxy-N-methyl-diphenylamine,2,7-diaminofluorene or triphenylamine.
 4. The method according to claim2, in which the enhancing agent is 4-amino-4'-methoxystilbene,4,4'-diaminostilbene-2,2'-disulfonic acid, iminostilbene,4,4'-dihydroxybenzophenone, N-benzylidene-4-biphenylamine,4,4'-diaminodiphenylamine, 4,4'-dimethoxy-N-methyl-diphenylamine,2,7-diaminofluorene or triphenylamine.
 5. A detergent additivecomprising an enzyme exhibiting laccase activity and an enhancing agentof the following formula; ##STR10## wherein E is a single bond, acarbonyl group or one of the following groups: (--CH═CH--)_(n),(--CH═N--)_(n) or (--NR¹¹ --) in which n represents an integer from 1 to2; andR¹ -R¹¹ may be identical or different and independently are one ofthe following radicals: hydrogen, hydroxy, formyl, carboxy or an esteror salt thereof, carbamoyl, sulfo or an ester or salt thereof,sulfamoyl, phospho, phosphono, phosphonooxy or an ester or salt thereof,nitro, amino, phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ - alkoxy, carbonyl-C₁ -C₅-alkyl, aryl-C₁ -C₅ -alkyl; wherein the carbamoyl, sulfamoyl, and aminogroups may be unsubstituted or substituted once or twice with asubstituent group R¹⁴ ; the phenyl group may be unsubstituted orsubstituted with one or more substituent groups R¹⁴ ; and the C₁ -C₁₄-alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkylgroups may be saturated or unsaturated, branched or unbranched, andunsubstituted or substituted with one or more substituent groups R¹⁴ ;wherein R¹⁴ is any of the following radicals; hydroxy, formyl, carboxyor an ester or salt thereof, carbamoyl, sulfo or an ester or saltthereof, sulfamoyl, phospho, phosphono, phosphonooxy or an ester or saltthereof, nitro, amino, phenyl, C₁ -C₅ -alkyl, C₁ -C₅ -alkoxy; whereinthe carbamoyl, sulfamoyl, and amino groups may be unsubstituted orsubstituted once or twice with hydroxy, C₁ -C₃ -alkyl, C₁ -C₃ -alkoxy;the phenyl group may be unsubstituted or substituted with one or more ofthe following radicals: hydroxy, amino, formyl, carboxy or an ester orsalt thereof, carbamoyl, sulfo or an ester or salt thereof, orsulfamoyl; and the C₁ -C₅ -alkyl and C₁ -C₅ -alkoxy groups may besaturated or unsaturated, branched or unbranched, and unsubstituted orsubstituted once or twice with any of the following radicals: hydroxy,amino, formyl, carboxy or an ester or salt thereof, carbamoyl, sulfo oran ester or salt thereof, or sulfamoyl; or in which R⁵ and R⁶ togetherform a group --B--, in which B is (--CH═N--)_(m), (--CH═CH--) or(--CHR¹⁴ --) in which m is 1 or 2 and R¹⁴ is as defined above.
 6. Thedetergent additive according to claim 4, in which the enhancing agent is4-amino-4'-methoxystilbene.
 7. The detergent additive according to claim4, in which the enhancing agent is 4,4'-diaminostilbene-2,2'-disulfonicacid.
 8. The detergent additive according to claim 4, in which theenhancing agent is iminostilbene.
 9. The detergent additive according toclaim 4, in which the enhancing agent is 4,4'-dihydroxybenzophenone. 10.The detergent additive according to claim 4, in which the enhancingagent is N-benzylidene-4-biphenylamine.
 11. The detergent additiveaccording to claim 4, in which the enhancing agent is4,4'-diaminodiphenylamine.
 12. The detergent additive according to claim4, in which the enhancing agent is4,4'-dimethoxy-N-methyl-diphenylamine.
 13. The detergent additiveaccording to claim 4, in which the enhancing agent is2,7-diaminofluorene.
 14. The detergent additive according to claim 5, inwhich the enhancing agent is triphenylamine.
 15. A detergent compositioncomprising a detergent additive according to claim 4 and a surfactant.16. The detergent composition according to claim 15, in which theenhancing agent is 4-amino-4'-methoxystilbene,4,4'-diaminostilbene-2,2'-disulfonic acid, iminostilbene,4,4'-dihydroxybenzophenone, N-benzylidene-4-biphenylamine,4,4'-diaminodiphenylamine, 4,4'-dimethoxy-N-methyl-diphenylamine,2,7-diaminofluorene or triphenylamine.
 17. A method of bleaching oflignin-containing material, comprising treating the material with anenzyme selected from the group consisting of laccase, catechol oxidase,monophenol monooxygenase and bilirubin oxidase and an enhancing agent ofthe following formula: ##STR11## wherein E is a single bond, a carbonylgroup or one of the following groups: (--CH═CH--)_(n), (--CH═N--)_(n) or(--NR¹¹ --), in which n represents an integer from 1 to 2; andR¹ -R¹¹may be identical or different and independently are one of the followingradicals: hydrogen, hydroxy, formyl, carboxy or an ester or saltthereof, carbamoyl, sulfo or an ester or salt thereof, sulfamoyl,phospho, phosphono, phosphonooxy or an ester or salt thereof, nitro,amino, phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ - alkoxy, carbonyl-C₁ -C₅ -alkyl,aryl-C₁ -C₅ -alkyl; wherein the carbamoyl, sulfamoyl, and amino groupsmay be unsubstituted or substituted once or twice with a substituentgroup R¹⁴ ; the phenyl group may be unsubstituted or substituted withone or more substituent groups R¹⁴ ; and the C₁ -C₁₄ -alkyl, C₁ -C₅-alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkyl groups may besaturated or unsaturated, branched or unbranched, and unsubstituted orsubstituted with one or more substituent groups R¹⁴ ; wherein R¹⁴ is anyof the following radicals; hydroxy, formyl, carboxy or an ester or saltthereof, carbamoyl, sulfo or an ester or salt thereof, sulfamoyl,phospho, phosphono, phosphonooxy or an ester or salt thereof, nitro,amino, phenyl, C₁ -C₅ -alkyl, C₁ -C₅ -alkoxy; wherein the carbamoyl,sulfamoyl, and amino groups may be unsubstituted or substituted once ortwice with hydroxy, C₁ -C₃ -alkyl, C₁ -C₃ -alkoxy; the phenyl group maybe unsubstituted or substituted with one or more of the followingradicals: hydroxy, amino, formyl, carboxy or an ester or salt thereof,carbamoyl, sulfo or an ester or salt thereof, or sulfamoyl; and the C₁-C₅ -alkyl and C₁ -C₅ -alkoxy groups may be saturated or unsaturated,branched or unbranched, and unsubstituted or substituted once or twicewith any of the following radicals: hydroxy, amino, formyl, carboxy oran ester or salt thereof, carbamoyl, sulfo or an ester or salt thereof,or sulfamoyl; or in which R⁵ and R⁶ together form a group --B--, inwhich B is (--CH═N--)_(m), (--CH═CH--) or (--CHR¹⁴ --) in which m is 1or 2 and R¹⁴ is as defined above.
 18. The method according to claim 17,in which the enzyme is a laccase.
 19. The method according to claim 17,in which the enhancing agent is 4-amino-4'-methoxystilbene,4,4'-diaminostilbene-2,2'-disulfonic acid, iminostilbene,4,4'-dihydroxybenzophenone, N-benzylidene-4-biphenylamine,4,4'-diaminodiphenylamine, 4,4'-dimethoxy-N-methyl-diphenylamine,2,7-diaminofluorene or triphenylamine.
 20. The method according to claim18, in which the enhancing agent is 4-amino-4'-methoxystilbene,4,4'-diaminostilbene-2,2'-disulfonic acid, iminostilbene,4,4'-dihydroxybenzophenone, N-benzylidene-4-biphenylamine,4,4'-diaminodiphenylamine, 4,4'-dimethoxy-N-methyl-diphenylamine,2,7-diaminofluorene or triphenylamine.
 21. A method of bleaching alignin-containing material, comprising treating the material with anenzyme selected from the group consisting of laccase, catechol oxidase,monophenol monooxygenase and bilirubin oxidase and an enhancing agent ofthe following formula: ##STR12## in which X is (--O--), (--S--), (--NR¹⁵--), (--CHR¹⁵ --) (>C═O), (--CH═CH--), (--CH═N--), or (--N═N--); and R¹-R⁹ and R¹⁵, which may be identical or different, independently are aradical selected from the group consisting of: hydrogen, hydroxy,formyl, carboxy and esters and salts thereof, carbamoyl, sulfo andesters and salts thereof, sulfamoyl, phospho, phosphono, phosphonooxyand esters and salts thereof, nitro, amino, phenyl, C₁ -C₁₄ -alkyl, C₁-C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ alkyl; in which thecarbamoyl, sulfamoyl, and amino groups may be unsubstituted orsubstituted once or twice with a substituent group R¹⁰ ; in which phenylmay be unsubstituted or substituted with one or more substituent groupsR¹⁰ ; and in which C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅-alkyl, and aryl-C₁ -C₅ -alkyl groups may be saturated or unsaturated,branched or unbranched, and may be unsubstituted or substituted with oneor more substituent groups R¹⁰ ; in which R¹⁰ independently represents aradical selected from the group consisting of hydroxy, formyl, carboxyand esters and salts thereof, carbamoyl, sulfo and esters thereof,nitro, amino, phenyl, aminoalkyl, piperidino, piperazinyl, pyrrolidino,C₁ -C₅ -alkyl, and C₁ -C₅ -alkoxy; in which the carbamoyl, sulfamoyl,and amino groups may be unsubstituted or substituted once or twice withhydroxy, C₁ -C₅ -alkyl, or C₁ -C₅ -alkoxy; in which phenyl may besubstituted with one or more radicals selected from the group consistingof hydroxy, amino, formyl, carboxy and esters and salts thereof,carbamoyl, sulfo and esters and salts thereof, and sulfamoyl; and inwhich C₁ -C₅ -alkyl and C₁ -C₅ -alkoxy groups may be saturated orunsaturated, branched or unbranched, and unsubstituted or substitutedonce or twice with any radicals selected from the group consisting ofhydroxy, amino, formyl, carboxy and esters and salts thereof, carbamoyl,sulfo and esters and salts thereof, and sulfamoyl; orin which two of R¹-R⁸ may together form a group --B-- which is selected from the groupconsisting of (--CHR¹⁰ --N═N--), (--CH═CH--)_(n), (--CH═N--)_(n) and(--N═CR¹⁰ --NR¹¹ --), in which n is an integer of from 1 to 3, R¹⁰ is asubstituent group as defined above and R¹¹ is defined as R¹⁰.
 22. Themethod according to claim 21, in which the enzyme is a laccase.
 23. Themethod according to claim 22, in which X is (--O--) or (--S--) and R⁵ isan alkyl group which may be further substituted.
 24. The methodaccording to claim 22, in which enhancing agent is10-methylphenothiazine, 10-phenothiazine-propionic acid,N-hydroxysuccinimide-10-phenothiazine-propionate,10-ethyl-4-phenothiazine-carboxylic acid, 10-ethylphenothiazine,10-propylphenothiazine, 10-isopropylphenothiazine,methyl-10-phenothiazinepropionate, 10-phenylphenothiazine,10-allylphenothiazine,10-(3-(4-methyl-1-piperazinyl)propyl)phenothiazine,10-(2-pyrrolidinoethyl)phenothiazine, 2-acetyl-10-methylphenothiazine,4-carboxy-10-phenothiazine-propionic acid, 10-methylphenoxazine,10-ethylphenoxazine, 10-phenoxazine-propionic acid,4-carboxy-10-phenoxazine-propionic acid,10-(2-hydroxyethyl)phenothiazine, 10-(2-hydroxyethyl)phenoxazine or10-(3-hydroxypropyl)phenothiazine.